Aims: We will apply our newly developed methods of microscopic lung imaging to determine cellular mechanisms of vectorial alveolo-capillary signaling serving lung immunity. We will model alveolar immune challenge through intra-alveolar infusions of tumor necrosis factor-alpha (TNF ) or lipopolysaccharide (LPS). We will determine alveolo-capillary signaling responses through quantifications in the alveolar epithelial cell (AEC), of the cytosolic and mitochondrial Ca2+ (Specific Aim), of reactive oxygen species (ROS) (Specific Aim 2), and of leukocyte recruitment in peri-alveolar capillaries (Specific Aim 3). These experiments will test the novel hypothesis that AEC ROS is the diffusible messenger in alveolo-capillary signaling. Procedures: The isolated, blood-perfused rat lung will be used. Intra-alveolar challenges will be instituted by alveolar micropuncture. AEC of intact alveoli will be imaged by conventional and confocal fluorescence microscopy. In situ quantifications will be obtained of (i) the cytosolic Ca2+ by the fura 2-ratio method, (ii) the mitochondrial Ca2+ using the fluorescent dye rhod 2, (iii) Ca2+ release from the endoplasmic reticulum using the dye fura-ff, and (iii) ROS productions using the H202-sensitive dye DCFH. cPLA2 activation and tyrosine phosphorylation will be assayed by in sit immunoimaging. Leukocyte recruitment in adjoining capillaries will be quantified in terms of the fluorescence of the dye rhodamine 6G. Significance: Host defense responses in alveolar innate immunity are attributable to the release of chemokines such as TNFalpha into the alveolar space. However, mechanisms by which alveolar chemokines convey proinflammatory signals to capillaries remain unclear. We previously identified that in AEC, increase of cytosolic Ca+2 and cPLA2 activation are critical for successful signaling. Our preliminary experiments indicate that ROS production is also critical. The proposed research will bring together these responses in a cogent and systematic understanding of vectorial alveolo-capillary signaling. No previous research addresses these issues. A novel understanding of lung immune processes will be achieved.